磷酸化Osteopontin抑制磷酸钙和草酸钙结晶的动力学研究

发布时间：2018-05-14 23:01

本文选题：磷酸化OPN多肽 + 磷酸钙　；参考：《华中农业大学》2015年博士论文

【摘要】：为了更好的理解植物体内草酸钙的生物矿化过程,本文借助模拟人体病理矿物(如肾结石)的形成,研究多肽分子对其形成的影响,从而比较植物与动物体内生物矿化作用的差异,为探明植物中草酸钙类晶体的生物矿化机理提供理论依据。人体肾结石中,含钙结石是最普遍的形式,约80%的肾结石由草酸钙和磷酸钙组成。尿液中的Osteopontin(OPN)蛋白可抑制草酸钙和磷酸钙的结晶。然而,对于OPN蛋白磷酸化程度和抑制效果之间关系,及OPN蛋白在纳米尺度调控矿物晶面台阶生长和溶解动力学机制的相关研究较少。本研究主要借助原位原子力显微镜,结合其他体相结晶技术及蛋白分析和表征手段,定量分析磷酸钙和草酸钙的成核及台阶生长和溶解动力学,得到的主要结果如下:1.OPN多肽羟基磷灰石(HAP)结晶的抑制与多肽磷酸化程度及浓度紧密相关在近生理条件下(p H 7.40,离子强度I=0.15 mol/L),借助电位监控的方法,研究含有14个氨基酸残基的OPN多肽(14-mer OPN)对HAP成核和生长的影响。结果发现,随着OPN多肽(156 nmol/L)磷酸化程度的增加,HAP成核所需的诱导时间显著延长。相同序列的非磷酸化OPN只在其浓度较高(234 nmol/L)时,才能通过延长诱导时间有效抑制HAP的成核。在一定浓度范围内(156 nmol/L),磷酸化修饰的OPN不仅提高了成核期磷酸钙纳米颗粒的稳定性,同时还抑制了纳米颗粒从无定形相向晶相的转变。这些表明,OPN多肽以磷酸化程度和浓度双重调控方式抑制HAP的结晶。2.磷酸化OPN多肽对二水磷酸氢钙(DCPD)(010)面台阶生长动力学和界面能借助原子力显微镜,原位探测磷酸化OPN多肽与DCPD(010)面的相互作用时发现,磷酸化OPN多肽专一地吸附[100]Cc方向台阶,从而抑制台阶的移动。经典的晶体生长Cabrera-Vermilyea物理模型可很好地解释在不同过饱和度下,磷酸化OPN多肽对台阶移动的抑制作用。此外,磷酸化OPN多肽侧链上的磷酸根通过静电作用,吸附在[100]Cc台阶改变矿物-水界面能,从而延迟晶面生长过程中台阶的形成。非磷酸化的OPN多肽完全不具备抑制效果。晶面生长动力学结果和体相成核结果一致。进一步证实了磷酸化OPN多肽对台阶动力学和界面能的双重控制作用。3.磷酸化OPN多肽显著抑制草酸钙的异质成核和团聚在模拟的酸性尿液生理条件下,借助原子力显微镜,原位观察尿液的两种组分草酸和OPN多肽对DCPD(010)面[101]Cc,[100]Cc和[101]Cc三个方向台阶溶解的影响。结果表明磷酸化OPN多肽专一性地抑制[101]Cc方向台阶的移动,进而减少钙离子的释放,并显著抑制DCPD表面诱导一水合草酸钙的成核。这不但体现了矿物溶解再结晶的过程,同时还展示了天然蛋白对此过程的调控作用,更加深了我们对结石形成抑制机制的理解。本文结合晶面动力学和体相结晶动力学的方法,着重探讨了多肽分子与矿物表面的相互作用。此研究不但揭示了磷酸钙-草酸钙病理矿化的动力学过程,同时还为理解香蕉体内生物矿化机制提供有效线索。
[Abstract]:In order to better understand the biological mineralization process of calcium oxalate in plants, this paper studies the effect of polypeptide molecules on its formation by simulating the formation of human pathological minerals, such as kidney stones, so as to compare the difference of biological mineralization in plants and animals, and provide a theoretical basis for exploring the mechanism of biomineralization of calcium oxalate crystals in plants. Calcium lithiasis is the most common form in human kidney stones. About 80% of the kidney stones are composed of calcium oxalate and calcium phosphate. The Osteopontin (OPN) protein in urine inhibits the crystallization of calcium oxalate and calcium phosphate. However, the relationship between the degree of phosphorylation and inhibition of OPN protein and the regulation of OPN in the nanometer scale regulation of mineral surface step birth In this study, the nucleation and step growth and dissolution kinetics of calcium phosphate and calcium oxalate were quantitatively analyzed by using in situ atomic force microscopy, other body phase crystallization techniques and protein analysis and characterization methods. The main results were as follows: the crystallization of 1.OPN polypeptide hydroxyapatite (HAP) crystallization The inhibition is closely related to the degree and concentration of polypeptides phosphorylation in near physiological conditions (P H 7.40, ionic strength I=0.15 mol/L). By means of potential monitoring, the effect of OPN polypeptide (14-mer OPN) containing 14 amino acid residues (14-mer OPN) on the nucleation and growth of HAP is studied. The results show that with the increase of the phosphorylation of OPN polypeptide (156 nmol/L), HAP formation is found. The non phosphorylated OPN of the same sequence can inhibit the nucleation of HAP by prolonged induction time only when its concentration is higher (234 nmol/L). In a certain concentration range (156 nmol/L), phosphorylated OPN not only improves the stability of calcium phosphate nanoparticles at the nucleation stage, but also inhibits the nano particles at the nucleation stage. The transformation of particles from amorphous phase to crystalline phase shows that OPN peptide inhibits the crystallization of HAP by the dual regulation of phosphorylation and concentration, and the interaction between.2. phosphorylated OPN polypeptide and OPN polyphosphate (DCPD) (DCPD) (DCPD) (010) surface step growth kinetics and interface can be used to detect the interaction of phosphorylated OPN polypeptide and DCPD (010) surface by atomic force microscope. It was found that the phosphorylated OPN polypeptide specifically adsorb the [100]Cc direction step, thus inhibiting the step movement. The classical crystal growth Cabrera-Vermilyea physical model can well explain the inhibition of the phosphorylated OPN polypeptide on the step movement under different supersaturation. In addition, the phosphate on the phosphorylated OPN polypeptide side chain is static by electrostatic action. The adsorption on the [100]Cc step changes the energy of the mineral water boundary, which delays the formation of the steps during the growth of the crystal surface. The non phosphorylated OPN polypeptide has no inhibitory effect. The crystal surface growth kinetics and the body phase nucleation result coincide. Further confirmed the dual control effect of the phosphorylated OPN polypeptide on the step dynamics and the interfacial energy of.3. phosphorus Acidified OPN polypeptide significantly inhibited the heterogenous nucleation and aggregation of calcium oxalate in the simulated acidic urine physiological conditions. The effects of two components of oxalic acid and OPN polypeptide on the dissolution of DCPD (010) surface [101]Cc, [100]Cc and [101]Cc in three directions were observed by atomic force microscopy. The results showed that the phosphorylated OPN polypeptide was specifically inhibited [ The movement of the 101]Cc direction step further reduces the release of calcium ions and significantly inhibits the nucleation of calcium oxalate hydrate on the surface of DCPD. This not only reflects the process of mineral dissolution and recrystallization, but also shows the regulation of the natural protein in this process, and further our understanding of the inhibition mechanism of the formation of stones. This article combines with the crystal surface. The kinetic and crystalline kinetics of the body phase are focused on the interaction between the peptide molecules and the mineral surface. This study not only reveals the kinetic process of calcium phosphate calcium oxalate, but also provides an effective clue to understand the mechanism of biomineralization in banana.

【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：Q945

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